Select Page

Comparative Physiology @ Guelph

Culture of mouse keratinocytes and feeder cells

Culture conditions: 32C, 5% CO2

 

Origin

Mouse keratinocytes from C57Bl6 strain.

 

Substrate

Keratinocytes grow optimally on feeder cells (e.g. 3T3J2 fibroblasts) or on collagen I-coated surfaces.

 

Thawing and plating cells

  • Thaw 1 freezing vial containing 2x106 cells (37°C water bath).
  • Transfer cells into 15 ml plastic tube containing 9 ml of pre-warmed medium and spin for 5 min at 1.000 rpm (at room temperature).
  • Re-suspend in 3 ml of FDA medium and plate on collagen-coated (5 μg/cm2) culture dish (3 cm diameter).
  • Change medium next day.

 

Subculture

  • To maintain their proliferative potential, Ca2+ must be depleted to 50 μM in the serum and must be absent from all solutions (e.g. PBS, DMEM etc…).
  • Passage cells only when they are confluent (density 5 to 6x104 cells/cm2).
  • Typical dilution is 1:2 (1:3 is the maximum).
  • Trypsinize cells with 0.1% trypsin/0.02% EDTA (5 min at 37°C) until cells detach.
  • Pellet the cells (1000 rpm for 4 minutes, RT).
  • Re-suspend cells in fresh medium and re-plate onto 2 dishes.
  • Replace medium every 2-3 days.

Freezing

2x106 cells/mL per vial (90 % FBS low Ca2+ and 10% DMSO).

 

Induction of terminal differentiation by addition of Ca2+

  • The next day after cells were seeded, change the medium from normal FAD media to FAD+ Medium containing 1.2 mM CaCl2.
  • Experiments should be performed 3 days after plating and 48-50h after the calcium shift.

 

NB: Experiments in low calcium conditions can be performed on the second day after plating.

 

Culture of keratinocyte Feeder cells

 

Origin

  • MEFs (3T3J2) fibroblasts.
  • SNLP (3T3J2) fibroblasts.

Subculture

  • Feeder cells grow in FDA medium, without insulin, cholera toxin and hydrocortisol.
  • Typical passage 1:6 (1:10 is maximum). Use 0.05% Trypsin/EDTA.
  • Before using them as feeders, cells must be inactivated by mitomycin C-treatment.

 

Mitomycin C-treatment

  1. Add 10 μg/ml mitomycin C to a confluent culture of feeder cells and place in incubator for 2-3 h.
  2. Wash dishes 5 times with PBS and 1 time with medium.
  3. Freeze cells (10cells/ml).
  4. Before using them, test they no longer divide for 4-5 days. Only use after this test has been performed successfully.

Keratinocytes feeding

  • Plate feeders (50.000 cells /cm2 = confluence) on collagen I-coated dishes 2h before plating keratinocytes, or together.

 

from Thomas Magin posted by Oualid Haddad 2013